PROJECT SUMMARY The Phase I of this program met or exceeded the success criteria, developing and implementing the capture free TempO-Seq assay, capable of measuring not just mRNA, but any nucleic acid target, verifying this platform with a whole human transcriptome assay and human surrogate transcriptome assay, both of which were launched as commercial products. The surrogate assay is a subset of genes from the whole transcriptome that is capable of identifying every known pathway, compound signatures, etc. without having to measure the whole transcriptome. The performance of these assays exceeded the success criteria and the original plans to implement assays of up to 1,000 mRNA targets, with the whole transcriptome assay targeting 30,247 RefSeq IDs (Affymetrix microarrays target only 30,654), and the surrogate measuring 2,733 genes. This Phase II falls under the NHGRI area of special interest Topic C: ?Genomics tools ranging from new instruments to sophisticated molecular biology kits?. It will extend the validation and cross- platform studies for the human assays, extend the assay to mouse by establishing a mouse whole transcriptome TempO-Seq assay, validate the accuracy with which the human and mouse surrogate assays identify pathways and predict gene changes across the whole transcriptome by testing diverse samples in the Hu and Mu TempO-Seq whole transcriptome assays, establish and validate focused sets of genes derived from the whole transcriptome archives that investigators can use as-is or selectively combine together or with the surrogate into a single assay customized for their research, implement a kit permitting sequencing on the Ion Torrent, and fully implement a TempO-SeqR analysis package that investigators can use to process their data and derive a file from which to derive dose and time course graphs, identify differentially expressed genes and false positive rates, identify expressed pathways, and predict (from the surrogate) what genes across the whole transcriptome that are not directly measured are expected to be differentially expressed, all without requiring a bioinformatics expert. The significance to investigators is that the TempO-Seq assays can provide the same coverage and as good or better sensitivity to measure rare transcripts as RNA-seq but 10 to 100 more samples can be sequenced for the same cost, which is important because more samples can be run to provide higher quality data and more detailed insights, or to profile more compounds, or more time points, or more patient samples. Investigators can make their budgets go further, and get faster turn-around time such as running whole transcriptome assays on the MiniSeq, an automated sequencer costing only $49,000, which otherwise cannot run whole transcriptome RNA-seq assays, and get higher quality data, without the bioinformatics expertise required for RNA-seq.